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EP2248907A1 - Gluco-oligosaccharides comprenant des liens (alpha 1-->4) et (alpha 1-->6) glycosidiquues, leur utilisation et procédés de fabrication - Google Patents

Gluco-oligosaccharides comprenant des liens (alpha 1-->4) et (alpha 1-->6) glycosidiquues, leur utilisation et procédés de fabrication Download PDF

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EP2248907A1
EP2248907A1 EP09159825A EP09159825A EP2248907A1 EP 2248907 A1 EP2248907 A1 EP 2248907A1 EP 09159825 A EP09159825 A EP 09159825A EP 09159825 A EP09159825 A EP 09159825A EP 2248907 A1 EP2248907 A1 EP 2248907A1
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Prior art keywords
gluco
oligosaccharide
glucosidic linkages
oligosaccharides
consecutive
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Rijksuniversiteit Groningen
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Rijksuniversiteit Groningen
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Priority to EP09159825A priority Critical patent/EP2248907A1/fr
Priority to CA2761257A priority patent/CA2761257C/fr
Priority to PCT/NL2010/050269 priority patent/WO2010128859A2/fr
Priority to JP2012509750A priority patent/JP5916602B2/ja
Priority to EP10722432.1A priority patent/EP2427565B1/fr
Priority to CN201080030033.0A priority patent/CN102459624B/zh
Priority to US13/319,237 priority patent/US9657322B2/en
Priority to MX2011011863A priority patent/MX2011011863A/es
Priority to ES10722432.1T priority patent/ES2444947T3/es
Priority to RU2011149793/10A priority patent/RU2571206C2/ru
Priority to BRPI1011572-2A priority patent/BRPI1011572A2/pt
Priority to AU2010245378A priority patent/AU2010245378B2/en
Publication of EP2248907A1 publication Critical patent/EP2248907A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01005—Dextransucrase (2.4.1.5)

Definitions

  • the invention relates to the field of poly- and oligosaccharides and their nutritional effects.
  • it relates to the application of ⁇ -glucanotransferases in methods for preparing dietary fibers, including prebiotic oligosaccharides, and to novel oligosaccharides obtainable thereby.
  • fibres are derived from carbohydrate sources that have a non-digestible component.
  • Fibres are typically divided into two categories; the insoluble fibres such as wheat bran, resistant starch, hemicelluloses, lignin etc., and the soluble fibres, which can be further classified into two subdivisions: short chain length soluble fibres, including polydextrose, inulin and oligosaccharides, and long chain length soluble fibres including pectins, gums (guar, locust bean, carrageenan, xanthan) and ⁇ -glucan (from oat or barley for example).
  • short chain length soluble fibres including polydextrose, inulin and oligosaccharides
  • long chain length soluble fibres including pectins, gums (guar, locust bean, carrageenan, xanthan) and ⁇ -glucan (from oat or barley for example).
  • Prebiotics are dietary fibres, as they are not digested by human enzymes but fermented by the flora of the large intestine. Thus they increase biomass and frequency of defecation, thus having a positive effect on constipation and on the health of the mucosa of the large intestine.
  • Prebiotic carbohydrates are naturally occurring and can be found in numerous foods, including asparagus, chicory, tomatoes and wheat, as well as being a natural component of breast milk.
  • prebiotic is a selectively fermented ingredient that allows specific changes both in the composition and/or activity in the gastrointestinal microflora that confers benefit upon host well-being and health
  • an ingredient is therefore required to (i) resist digestion (gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption); (ii) be fermented by the gastrointestinal microbiota; and (iii) selectively stimulate the growth and/or activity of intestinal bacteria associated with health and well-being.
  • Prebiotics are generally recognised for their ability to alter the colonic microbiota, promoting a healthier composition and/or activity by increasing the prevalence of saccharolytic (carbohydrate fermenting) micro-organisms while reducing putrefactive (protein fermenting) micro-organisms.
  • Established non-digestible carbohydrates that fulfil the prebiotic criteria include fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose, inulin and polydextrose.
  • FOS fructo-oligosaccharides
  • GOS galacto-oligosaccharides
  • lactulose inulin and polydextrose.
  • Polydextrose is a polysaccharide composed of randomly cross-linked glucose units with all types of glycosidic bonding. Litesse polydextrose is resistant to digestion due to its unique arrangement of glycosidic linkages. Molecularly, ( ⁇ 1 ⁇ 6) bonds predominate, but about 13% of the polymer has ( ⁇ 1 ⁇ 4) linkages, which can be hydrolyzed by enzymes in the human small intestine. It is fermented throughout the colon and is particularly efficient at mediating a prebiotic effect in the distal colon. Human intervention studies have demonstrated that Litesse polydextrose enhances both bifidobacteria and lactobacilli in a dose dependent manner.
  • Starch is a polysaccharide found commonly in green plants - those containing chlorophyll - as a means of storing energy. Starch forms an integral part of the multi-billion food ingredients market and is characterised by its complex and consolidated nature. Starch is an ideal example of an essential commodity with a wide array of industrial applications, which include paper and card-board making, fermentation, biofuels, biodegradable plastics and detergents, bio-pesticides, surfactants, polyurethane, resins, binders and solvents. However, it is the food industry that provides the largest market for starch and its derivatives.
  • Starch is either degraded completely in the small intestine to glucose and taken up in the blood or those parts that escape digestion end up in the large intestine where they serves as a general substrate for the colonic microbial flora. Starch and its derivatives in itself do not stimulate specific beneficial colon microbes. Thus, starch in itself is not a prebiotic compound.
  • the partial solution to the problem is to degrade starch into the disaccharide maltose and then use a transglucosidase enzyme to convert the maltose into ( ⁇ 6)-linked isomalto-oligosaccharides (IMO) with a degree of polymerization of 2 to 4. These IMO products are, however, too short and are mostly degraded in the small intestine, thus not reaching the colon.
  • That part of the IMO product that reaches the colon is quickly degraded in the proximal part of the colon by the intestinal microflora and does not reach the distal part were more malign, protein degrading bacteria reside.
  • protein degrading bacteria reside.
  • beneficial bacterial species in particular bifidobacteria, longer isomalto-oligosaccharides are required.
  • the present invention provides further means and methods for the (enzymatic) modification of starch, starch derivatives and/or MOS of different chain length in order to change their functional properties and enhance their nutritional value.
  • GTFB gluco-oligosaccharides comprising ( ⁇ 1 ⁇ 4) linked glucose residues, such as malto-oligosaccharides (MOS).
  • MOS malto-oligosaccharides
  • GTFB activity can thus yield a series of linear gluco-oligosaccharides up to at least DP35.
  • Structural analysis of the products has revealed that GTFB cleaves ( ⁇ 1 ⁇ 4) glucosidic bonds and makes new ( ⁇ 1 ⁇ 4) and ( ⁇ 6) bonds.
  • this enzyme is designated as (1 ⁇ 4)- ⁇ -D-glucan: (1 ⁇ 4) (1 ⁇ 6)- ⁇ -D-glucan ⁇ -D-glucanotransferase or, alternatively, as ⁇ -glucanotransferase.
  • Glucansucrase enzymes also use MOS but only as acceptor substrates in the presence of sucrose as donor substrate.
  • the products that GTFB can synthesize from starch or starch derivatives contain relatively long isomalto-oligosaccharide (IMO) side chains, in particular IMO side chains with a degree of polymerization of 4 and higher.
  • IMO-MALT isomalto-oligosaccharide
  • IMO-MALT can therefore be considered as a partially resistant starch derivative giving less glucose production in the small intestine than unmodified maltodextrin would give. This is considered beneficial and contributes to a healthy life style (reduce the risk of developing obesity, type II diabetes, and heart and coronary diseases related to the overconsumption of quickly degradable starch/derivatives).
  • the IMO-MALT part that passes unmodified into the colon will be further degraded by the residual microflora.
  • the IMO part of the IMO-MALT containing ( ⁇ 1 ⁇ 6) linkages can act as a specific substrate for beneficial bifidobacteria, making IMO-MALT a prebiotic ingredient.
  • IMO-MALT therefore has at least the following benefits:
  • the invention relates to a method for producing a mixture of gluco-oligosaccharides having one or more ( ⁇ 1 ⁇ 6) glucosidic linkages and one or more ( ⁇ 1 ⁇ 4) glucosidic linkages, comprising contacting a poly- and/or oligosaccharide substrate comprising at its non-reducing end at least two ( ⁇ 1 ⁇ 4)-linked D-glucose units with an ⁇ -glucanotransferase enzyme capable of cleaving ( ⁇ 1 ⁇ 4) glucosidic linkages and making new ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) glucosidic linkages.
  • the ⁇ -glucanotransferase is capable of transferring a maltosyl-, a maltotriosyl- or a maltotetraosyl-unit to the substrate via a new ( ⁇ 1 ⁇ 6) glucosidic linkage.
  • the gluco-oligosaccharide product(s) are linear or contain linear stretches of primarily ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) glucosidic linkages, rendering them resistant to enzymatic attack in the small intestine.
  • the ⁇ -glucanotransferase preferably does not introduce ( ⁇ 1 ⁇ 6) branching points, ( ⁇ 1 ⁇ 2) nor ( ⁇ 1 ⁇ 3) linkages.
  • the ⁇ -glucanotransferase is a new member of the GH70 family of glucansucrases [http://www.cazy.org], or a functional homolog thereof having the specified enzymatic activity and substrate preference as described above.
  • the enzyme is selected from the group consisting of GTFB from Lactobacillus reuteri 121, GTF106B from Lactobacillus reuteri TMW 1.106, GTML4 from Lactobacillus reuteri ML1 and GTFDSM from Lactobacillus reuteri DSM 20016 A , GTF from Lactobacillus fermentum ATCC 14931, all of which are known in the art both at the protein and nucleic acid level.
  • a GTFB homolog is used that shows at least 55%, preferably at least 60%, 75%, like at least 80%, 85%, or at least 90%, sequence identity at the amino acid level with GTFB from Lactobacillus reuteran 121 (AAU08014), GTF106B from Lactobacillus reuteri TMW 1.106 (ABP88725), GTML4 from Lactobacillus reuteri ML1 (AAU08003), GTFDSM from Lactobacillus reuteri DSM 20016 A (ABQ83597) or GTF from Lactobacillus fermentum ATCC 14931 (ZP_03945763).
  • a GTFB homolog is used that shows at least 55%, preferably at least 60%, 75%, like at least 80%, 85%, or at least 90%, sequence identity at the amino acid level with GTFB from Lactobacillus reuteri 121.
  • the enzyme shows at least 45%, more preferably at least 50%, sequence identity or at least 60% sequence identity at the amino acid level with the catalytic core of GTFB, the catalytic core being represented by the contiguous amino acid sequence W 790 YRP....IVMNQ 1484 as found in the protein sequence of GFTB of L. reuteri 121: GenBank accession number AAU08014 (protein code).
  • the homolog preferably comprises one or more of the following conserved amino acid residues, wherein the numbering corresponds to the position in GTFB of Lactobacillus reuteri 121 : Arg1013; Asp1015; Ala1017; Asn1019; Glu1053, Gly1054, Tyr1055, His1124, Asp1125, Gln1126, Arg1127, Lys1128, Asp1479; Ile1480, Met1482, Asn1483, Gln1484.
  • at least the catalytic residues Asp1015, Glu1053 and Asp1125 are present. More preferably, all of these residues are present.
  • the enzyme comprises at least one of the following consensus sequences wherein the numbering corresponds to the amino acid position in GTFB (see Figure 1 ):
  • the enzyme is a GTFA member from the glucansucrase group, for instance GTFA from Lactobacillus reuteri 121 (GenBank accession number AX306822 or AY697435 (GTF sequence + flanking sequences a.o. GTFB + transposases) , that has been genetically engineered to obtain the unique "GTFB-like" substrate specificity and activity required for practicing a method of the present invention.
  • GTFA member from the glucansucrase group for instance GTFA from Lactobacillus reuteri 121 (GenBank accession number AX306822 or AY697435 (GTF sequence + flanking sequences a.o. GTFB + transposases) , that has been genetically engineered to obtain the unique "GTFB-like" substrate specificity and activity required for practicing a method of the present invention.
  • the invention thus also relates to a genetically modified enzyme belonging to the gtfA type of glucansucrase enzymes comprising at least one of the mutations of Table 1, said enzyme being capable of cleaving ( ⁇ 1 ⁇ 4)glucosidic linkages and making new ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) glucosidic linkages and having a substrate preference for poly- and/or oligosaccharide substrates comprising ( ⁇ 1 ⁇ 4)-linked D-glucose units, in particular malto-oligosaccharides.
  • mutations equivalent to those mentioned in Table 1 can be introduced in GTFA enzyme homologues from other organisms.
  • GTF180 from Lactobacillus reuteri 180 GTFML1 from Lactobacillus reuteri ML1, DSRS from Leuconostoc mesenteroides B512-F, GTFD from Streptococcus mutans GS-5 (also see van Hijum et al 2006).
  • multiple mutations selected from Table 1 are introduced. In a specific embodiment, all positions shown in Table 1 are altered.
  • the enzyme is a GTFB type of glucansucrase, for example selected from the group consisting of GTFB from Lactobacillus reuteri 121, GTF106B from Lactobacillus reuteri TMW 1.106, GTML4 from Lactobacillus reuteri ML1, GTFDSM from Lactobacillus reuteri DSM 20016 A or GTF from Lactobacillus fermentum ATCC 14931, or a natural or artificial homolog (mutant) thereof.
  • the enzyme is GTFB from Lactobacillus reuteri 121.
  • a temperature of at least 30°C, preferably 37°C is used. It may be desirable, for instance in view of substrate properties and/or sterility, to work at an elevated temperature, like at least 70°C, provided that the enzyme is sufficiently heat stable.
  • the dry matter content of the reaction mixture can vary. In one embodiment, it is at least 10%, preferably at least 25%.
  • oligosaccharide substrates or substrate mixtures can be used in a method according to the invention, provided that they comprises poly and/or oligosaccharides whose non-reducing end contains ( ⁇ 1 ⁇ 4) linked glucose residues.
  • said non-reducing end contains 3 or more consecutive ( ⁇ 1 ⁇ 4)-linked glucose residues. Linear substrates are preferred.
  • a method for producing a mixture of linear gluco-oligosaccharides having one or more ( ⁇ 1 ⁇ 6) glucosidic linkages and one or more ( ⁇ 1 ⁇ 4) glucosidic linkages comprising contacting a linear poly- and/or oligosaccharide substrate comprising at its non-reducing end at least two ( ⁇ 1 ⁇ 4)-linked D-glucose units with an ⁇ -glucanotransferase enzyme capable of cleaving ( ⁇ 1 ⁇ 4) glucosidic linkages and making new ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) glucosidic linkages.
  • the substrate has a degree of polymerization of at least 4, preferably at least 5, more preferably at least 6.
  • the substrate is for instance selected from the group consisting of native starch, modified starch, starch-derivatives, malto-oligosaccharides, amylose, amylopectin, maltodextrins, ( ⁇ 1 ⁇ 4) glucans, reuteran, or combinations thereof.
  • starch derivative refers to the product of native starch that has undergone one or more modifications, be it by physical and/or (bio)chemical means. Modifications include depolymerization, cross linking and substitution.
  • the starch or starch derivative can originate from various plant sources, including potato, maize, tapioca or wheat.
  • Some of the other raw materials include; rice, cassava, arrowroot, mung bean, peas, barley, oats, buckwheat, banana, sorghum and lentils.
  • Starch (derivative) from potato, maize, tapioca or wheat is preferred.
  • a method of the invention uses amylomaltase (AMase)-treated starch (ATS), preferably potato starch, as substrate.
  • AMase amylomaltase
  • ATS is commercially available from AVEBE (Veendam The Netherlands) under the trade name EteniaTM.
  • a further specific embodiment employs reuteran as substrate, which is an ⁇ -glucan product of reuteransucrase activity and comprises ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) linkages. Also a mixture of reuteran and malto-oligosaccharides (MOS) yields very good results.
  • reuteran as substrate, which is an ⁇ -glucan product of reuteransucrase activity and comprises ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) linkages.
  • MOS malto-oligosaccharides
  • a method of the invention as described above will typically yield a mixture of various linear gluco-oligosaccharides having one or more consecutive ( ⁇ 1 ⁇ 6) glucosidic linkages and one or more consecutive ( ⁇ 1 ⁇ 4) glucosidic linkages.
  • the mixture can essentially be used as such and does not require further purification.
  • various methods known in the art can be used, for example precipitation-fractionation or chromatography techniques.
  • a method of the invention comprises subjecting the mixture to size exclusion and/or anion exchange chromatography and isolating at least one gluco-oligosaccharide having one or more ( ⁇ 1 ⁇ 6) glucosidic linkages and one or more ( ⁇ 1 ⁇ 4) glucosidic linkages.
  • an enzyme activity as disclosed herein can give rise to an oligosaccharide with a unique structure.
  • the linear (i.e. unbranched) gluco-oligosaccharide of the general formula A-B, or the mixture comprising different gluco-oligosaccharides of the general formula A-B characterized in that (i) the linkage between the moiety A and the moiety B is an ( ⁇ 1 ⁇ 6) glucosidic linkage, (ii) moiety A comprises two or more consecutive ( ⁇ 1 ⁇ 6) glucosidic linkages, preferably wherein A comprises an isomalto-oligosaccharide with a degree of polymerization of at least 4 glucose residues and (iii) B comprises at least two, preferably at least three, consecutive ( ⁇ 1 ⁇ 4) linked glucose residues.
  • the A moiety consists of a series of consecutive ( ⁇ 1 ⁇ 6) linked glucose residues and the B moiety consists of a series of consecutive ( ⁇ 1 ⁇ 4) linked glucose residues.
  • the A moiety comprises one or more consecutive ( ⁇ 1 ⁇ 4) glucosidic linkages, preferably wherein A comprises a malto-oligosaccharide with at least four ( ⁇ 1 ⁇ 4) linked glucose residues.
  • a stretch of ( ⁇ 1 ⁇ 4) linked residues can be linked via an ( ⁇ 1 ⁇ 6) linkage to another stretch of ( ⁇ 1 ⁇ 4) linked residues.
  • the gluco-oligosaccharide has a degree of polymerization (DP) of at least 7 (DP ⁇ 7), preferably at least 10 (DP ⁇ 10), more preferably at least 15. Oligosaccharides with a length up to more than 30 residues have been observed according to MALDI-TOF-MS analysis.
  • DP degree of polymerization
  • the relative amount of the high molecular mass products DP10-DP35 in a mixture is less than the amount of products DP ⁇ 10.
  • An exemplary mixture has an average degree of polymerization of at least 5, preferably at least 6, such as between 6 and 15.
  • malto-oligosaccharides e.g. DP7, or DP6
  • a series of linear gluco-oligosaccharides are produced, and often different structures of a given DP are observed. For instance, at least 4 DP8 structures were identified, each differing with respect to the number of ( ⁇ 1 ⁇ 6) and ( ⁇ 1 ⁇ 4) glucosidic linkages. See also Figure 4 .
  • the ratio ( ⁇ 1 ⁇ 6) to ( ⁇ 1 ⁇ 4) glucosidic linkages and the structural diversity increases with increasing chain length.
  • At least 20%, preferably at least 25%, of the linkages is ( ⁇ 1 ⁇ 6).
  • the ratio between ( ⁇ 1 ⁇ 6) and ( ⁇ 1 ⁇ 4) glucosidic linkages generally ranges between 20:80 and 90:10.
  • a linear DP7 product with two consecutive ( ⁇ 1 ⁇ 6) linkages and four consecutive ( ⁇ 1 ⁇ 4) linkages
  • linear DP8 product with two consecutive ( ⁇ 1 ⁇ 6) linkages and five consecutive ( ⁇ 1 ⁇ 4) linkages
  • a DP10 with five consecutive ( ⁇ 1 ⁇ 6) linkages and four consecutive ( ⁇ 1 ⁇ 4) linkages.
  • the oligosaccharide (mixture) preferably comprises substantial amounts of both ( ⁇ 1 ⁇ 6) and ( ⁇ 1 ⁇ 4) glucosidic linkages. Therefore, in one embodiment the ratio between ( ⁇ 1 ⁇ 6) and ( ⁇ 1 ⁇ 4) glucosidic linkages is between 30:70 and 70:30.
  • a gluco-oligosaccharide or gluco-oligosaccharide mixture according to the invention has important industrial applications, in particular in nutritional and dietary compositions.
  • a (human or animal) food product comprising a gluco-oligosaccharide or gluco-oligosaccharide (mixture) according to the invention.
  • the food product can be a solid, semi-solid or liquid food product.
  • the food product can be a conventional nutritional product or a dietetic product. It can be a ready-to-eat food item or a food product that requires further handling prior to consumption, like a bake-off bread product.
  • Exemplary products include a dairy product, baby or infant formula, bakery product, pasta product, noodle product, confectionery product, liquid drink, sport drink, beverage and ice cream.
  • a further embodiment relates to the use of a gluco-oligosaccharide or gluco-oligosaccharide mixture according to the invention as food additive, for example as prebiotic fiber.
  • Prebiotics can be used in multiple food applications from dairy through to bakery, confectionery and beverage applications. Due to their chemical and physical structure they tend to be highly soluble and have the ability to improve body, texture and mouth feel.
  • Another useful application relates to inhibiting enzymes of the alpha-amylase type, such as salivary and pancreatic amylases. These enzymes normally act on a ( ⁇ 1 ⁇ 4) malto-oligosaccharide chain with DP ranging from 4-6. It is hypothesized that the presence of (non-hydrolyzable) ( ⁇ 1 ⁇ 6) linkages in an oligosaccharide of the invention only results in enzyme binding but not in glucose release. Addition of such oligosaccharides would lower the rate of metabolism of (e.g. starch metabolism), thereby reducing the glycaemic index (GI) of a food product.
  • GI glycaemic index
  • a gluco-oligosaccharide (mixture) according to the invention can therefore also help to reduce caloric value and/or the glycaemic load of food products. It thus contributes to a low GI diet. This is of particular interest for human health in general as well as in specific metabolic diseases, including diabetes mellitus and obesity.
  • the glucooligosaccharide finds its use in a therapeutical or cosmetic application, in particular for controlling a normal skin flora and promoting a healthy skin.
  • the oligosaccharide can bring about a probiotic effect in that it can preferably be utilized selectively by saprophytic bacteria.
  • the oligosaccharide can promote the growth of beneficial skin bacteria (e.g. Micrococcus kristinae ) compared to the growth of less desirable bacteria such as Staphylococcus aureus and Corynebacterium xerosis.
  • a cosmetic compositions comprising a gluco-oligosaccharide or gluco-oligosaccharide mixture according to the invention and a suitable carrier. It is also possible to employ the glucooligosaccharide (mixture) in a personal care item, for instance to include an absorbent article such as a disposal diaper, sanitary napkin, or the like which can reduce odor and dermatitis (rash) generated when such an absorbent article is worn.
  • an absorbent article such as a disposal diaper, sanitary napkin, or the like which can reduce odor and dermatitis (rash) generated when such an absorbent article is worn.
  • Glucansucrase (GS) (or glucosyltransferase; GTF) enzymes (EC 2.4.1.5) of lactic acid bacteria (LAB) use sucrose to synthesize a diversity of ⁇ -glucans with ( ⁇ 1 ⁇ 6) [dextran, mainly found in Leuconostoc], ( ⁇ 1 ⁇ 3) [mutan, mainly found in Streptococcus ], alternating ( ⁇ 1 ⁇ 3) and ( ⁇ 1 ⁇ 6) [alternan, only reported in Leuconostoc mesenteroides ], ( ⁇ 1 ⁇ 4) [reuteran, by GTFA and GTFO from Lactobacillus reuteri strains] glucosidic bonds ⁇ Monchois, 1999; van Hijum, 2006; Arguello-Morales, 2000 ;Kralj, 2002 ;Kralj, 2005 ⁇ .
  • Lactobacillus reuteri 121 uses the glucansucrase GTFA and sucrose as substrate to synthesize a reuteran product with large amounts of ( ⁇ 1 ⁇ 4) glucosidic linkages. Upstream of this gtfA gene another putative glucansucrase gene was identified designated gtfB. Previously it has been shown that after cloning and expression of this gene the enzyme showed no activity on sucrose as substrate. Also in the genome of L. reuteri ML1 the putative catalytic and C-terminal domain of a gtfB homolog, gtfML4, was identified upstream of gtfML1 encoding a mutansucrase ⁇ Kralj, 2004 ⁇ .
  • L. reuteri DSM 20016 also a GTFB homolog could be identified (73% identity 85% similarity in 883 amino acids). Furthermore, also L. reuteri TMW1.106 contains besides a GTFA homolog (GTFA106) a GTFB homolog (GTFB106). This enzyme showed 92% identity and 95% similarity in 1383 amino acids with GTFB from L. reuteri 121. However, in contrast to GTFB, GTF106B showed low (after 27 h of incubation) hydrolyzing activity on sucrose ⁇ Kaditzky, 2008 ⁇ .
  • GTFA106 GTFA homolog
  • GTFB106 GTFB homolog
  • GTFB has a disproportionation type and polymerizing type of activity on malto-oligosaccharides.
  • the enzyme uses malto-oligosaccharides (containing only ( ⁇ 1 ⁇ 4) glucosidic linkages) as substrate to synthesize oligosaccharides up to a degree of polymerization (DP) of 35.
  • DP degree of polymerization
  • E coli strains Bacterial strains, plasmids, media and growth conditions.
  • Escherichia coli TOP 10 (Invitrogen, Carlsbad, Calif.) was used as host for cloning purposes.
  • Plasmids pET15b (Novagen, Madison, WI) was used for expression of the (mutant) gtfB genes in E . coli BL21 Star (DE3). (Invitrogen).
  • E . coli strains were grown aerobically at 37 °C in LB medium ⁇ Ausubel, 1987 ⁇ .
  • E . coli strains containing recombinant plasmids were cultivated in LB medium with 100 ⁇ g ml -1 ampicillin. Agar plates were made by adding 1.5% agar to the LB medium.
  • plasmids Construction of plasmids .
  • Appropriate primer pairs and template DNA were used to create two different expression constructs with a C-terminal His-tag: for the complete GTFB (1587 amino acids), constructed using three separate PCR reactions using the method previously described for GTFA from Lb. reuteri 121 (see below) ⁇ Kralj, 2002 ⁇ , and an N-terminally truncated variant (without N-terminal) variable region of GTFB (889 amino acids).
  • gtfB was divided and cloned in three parts.
  • the first of the two Pst I restriction sites (1385 bp, 1751 bp) was altered, using the megaprimer method ⁇ Sarkar, 1990 ⁇ and the following primers: BpstIfor 5'-GTAAGTCGTTACTCAGCAGATGCTAATGG-3' containing a mutated Pst I restriction site (underlined, silent mutation by change of base shown in bold face), and, BpstI rev 5'-GGTCAGTAAATCCACCGTTATTAATTGG-3'.
  • the amplified product (420 bp) was used as (reverse) primer together with Bfor: 5'-GCAATT GTCGA CCATGG ATACAAATACTGGTGATCAGCAAACTGAACA-GG-3' containing Sal I (italics) and Nco I (bold) restriction sites.
  • the resulting product of 1700 bp was digested with Sal I and Pst I and ligated in the corresponding sites of pBluescript II SK + , yielding pBSP1600.
  • the amplified 420 bp product was also used as a forward primer together with BrevBamHI 5'-GGACTGTTATCACTATTATTATTTCCGGCC-3' 70 bp downstream of a Bam HI restriction site.
  • the resulting product of ( ⁇ 1500 bp) was digested with Pst I and Bam HI and ligated in the corresponding sites of pBluescript II SK + , yielding pBPB1000.
  • the third fragment was obtained using primers BforBamHI 5'-CGCTATGTAATTGAACAGAGTATTGCTGC-3' 200 bp downstream of a Bam HI restriction site and BRevHis 5'-CCTCCTT TCT AGA TCT ATTA GTGATGGTGATGGTGATG GTTGTTAAAGTTTAATG AAATTGCAGTTGG-3' containing Xba I (italics) and Bgl I (bold) and a 6 ⁇ histidine tag (underlined).
  • the resulting product of 2300 bp was digested with Bam HI and Xba I and ligated in the corresponding sites of pBluescript II SK+, yielding pBBX2300.
  • the complete gene was assembled as follows: pBPB1000 was digested with Pst I and Bam HI and the resulting fragment was ligated into pBSP1600 restricted with the same restriction enzymes yielding pBSB2600 (containing the first and second fragment).
  • plasmid pBBX2300 was digested with Bam HI and Sac II (present on the plasmid, used instead of Xba I) and the fragment was ligated into pBSB2600 yielding pBSS4900 containing the full length gtfB gene.
  • This plasmid was digested with Nco I and Bgl II and the gtfB gene was ligated in the Nco I and Bam HI sites of pET15b, yielding pET15B-GTFB.
  • GTFB GTFB Expression and purification of GTFB .
  • An overnight culture of E. coli BL21star (DE3) harbouring (mutant) GTFB ⁇ Kralj, 2004 ⁇ was diluted 1/100. Cells were grown to OD 600 0,4 and induced with 0.2 mM IPTG, after 4 h of growth cells were harvested by centrifugation (10 min at 4 °C at 10,000 ⁇ g ). Proteins were extracted by sonication and purified by Ni-NTA and anion exchange chromatography as described previously for the GTFA (reuteransucrase) from Lactobacillus reuteri 121 ⁇ Kralj, 2004 ⁇ , with the following modification: for anion exchange chromatography a 1 ml Hi-trapTM Q HP colum was used (Ge Healthcare).
  • GTFB is the first representative of a group of homologues enzymes identified in different Lactobacilli. Alignments of members of this novel group of enzymes with other glucansucrases showed similarities but also some characteristics differences.
  • region II encompassing the putative nucleophilic residue
  • region III the region downstream of the putative acid/base catalyst E1061 is completely different between the glucansucrases and the ⁇ -glucanotransferases.
  • region II encompassing the putative nucleophilic residue
  • region III the region downstream of the putative acid/base catalyst E1061 is completely different between the glucansucrases and the ⁇ -glucanotransferases.
  • the conserved tryptophan 1063 is substituted by a tyrosine residue in the ⁇ -glucanotransferases ( Fig. 1 ).
  • region IV the GTFB homologues contain a gap immediately upstream of the location of the Q1137 residue and at the position of the conserved glutamine a lysine residue is present.
  • the full length, N-terminal truncated version and putative nucleophilic mutant of GTFB were constructed and expressed successfully. Both the full length as well as the N-terminal truncated variant showed clear activity on malto-oligosacharides as measured by TLC (data not shown).
  • the constructed truncated GTFB version (GTFB- ⁇ N) was not expressed as efficiently as the full length GTFB and therefore all experiments were performed using full length GTFB.
  • an empty pET15b plasmid was purified, and already after His-tag purification no activity on malto-oligosaccharides (G2-G7) was detected (data not shown).
  • the purified full length D1015N (putative) nucleophilic mutant showed no activity on malto-oligosaccharides (G2-G7; data not shown).
  • the optimal activity for GTFB with maltotetraose as a substrate as determined qualitatively by TLC was at a temperature of 30-37 °C and a pH of 4-5 (data not shown). Combinations of different temperatures and pH buffers indicated optimal activity at a temperature of 37 °C and a pH of 4.7, which was used in all subsequent assays.
  • oligosaccharides Especially on malto-oligosaccharides with a degree of polymerization of 4 and larger, different oligosaccharides were synthesized ( Fig. 2 ). From a DP of 6 and larger, besides oligosaccharides also larger polymeric material started to accumulate. On amylose-V (Avebe, Foxhol, The Netherlands) also low activity (mainly G1 and G2 release) was observed ( Fig. 4 ). On maltose alone virtually no activity was observed. However, when amylose-V was incubated simultaneously with glucose or maltose a range of oligosaccharides were synthesized ( Fig. 4 ).
  • the first reaction products detectable on G6 were G1 (glucose) and G5 (maltopentaose) ( Fig. 3 ). Also on G7 the first products released were G1 (glucose) and G6 (maltohexaose). Later in time on G6 also other malto-oligosaccharides such as G2, G3 and G4 appeared. Also unknown saccharides next to G7 and G8 were identified, which besides ( ⁇ 1 ⁇ 4) glucosidic linkages also must contain other linkages indicated by the shift in their retention time.
  • region III and IV control the product specificity of GTF enzymes regarding the glycosidic bonding type formed.
  • region I and region II contain amino acid residues that contribute to enzyme activity and reaction specificity.
  • the amino acid residues of the conserved sequence regions form part of the acceptor substrate binding region of GTF enzymes.
  • sucrose As substrate these residues interact with the glucose (subsite-1) and fructose (subsite +1) moiety of sucrose.
  • GTFB utilizes maltoheptaose (and other malto-oligosaccharides) as substrate a glucose moiety will interact at the acceptor subsites in the GTFB enzyme.
  • the 3D structure of GTF180 shows some additional residues interacting at subsites -1 and +1 that are likely important for the interconversion of reaction specificity of GTFs.
  • the 981 L ⁇ V mutations is based on an interaction seen in the 3D structure of GTF180, where Leu981 has a Van Der Waals interaction with the fructosyl moiety of sucrose. In GTFB this position is occupied by a valine residue as well as in the other ⁇ -glucanotransferases GTFDSM, GTF106B, GTFML4.
  • the 1463 D ⁇ R, T or M mutations are aimed at substituting the aspartate residue, which is highly conserved in glucansucrases, but not in GTFB and the related enzymes, and interacts with the glucose moiety at subsite -1 in GTF180 3D structure.
  • the mutations may be combined in any manner to obtain a stronger effect in alteration of the reaction specificity GTF enzymes.
  • the proposed mutations are as follows:

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ES2444947T3 (es) 2014-02-27
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RU2011149793A (ru) 2013-06-20
CN102459624A (zh) 2012-05-16
AU2010245378A1 (en) 2011-12-01
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